A new sensitive LC/MS/MS analysis of Vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine

Debin Wan, Jun Yang, Bogdan Barnych, Sung Hee Hwang, Kin Sing Stephen Lee, Yongliang Cui, Jun Niu, Mitchell Aaron Watsky, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


There is an increased demand for comprehensive analysis of Vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyVitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, Vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the Vitamin D derivatives. This precludes a survey of numerous Vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting Vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2-to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying Vitamin D metabolites in a smaller sample volume (100 μL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

Original languageEnglish (US)
Pages (from-to)798-808
Number of pages11
JournalJournal of Lipid Research
Issue number4
StatePublished - 2017


  • 1,25-dihydroxyVitamin D
  • Major Vitamin D metabolites
  • Matrix effect
  • Quantification
  • Smaller sample volume

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology


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